Various chromatography techniques are known in the art for purifying proteins. Procedures such as molecular sieve chromatography, ion exchange chromatography, and electrophoresis are commonly utilized to purify proteins. Separation of proteins that have very similar molecular weights and similar net charges, however, requires the use of alternative purification methods due to the absence of any significant differential in the features (i.e., molecular weight and net charge) which known separation processes exploit. Complete and efficient separation of proteins intended for therapeutic use is critical, particularly if the purified protein is to be used in the treatment of hypersensitive individuals such as immunodeficient or immunocompromised patients.
An alternative technique for purifying proteins under limited conditions has been named "Immobilized Metal Affinity Chromatography" (IMAC). The development of this method resulted from the recognition that certain proteins have an affinity for heavy metal ions, which could be an additional distinguishing feature to use in attempting separation of the proteins. This feature applies especially to proteins containing histidine or cysteine residues, which have been found to complex with chelated zinc or copper ions and become adsorbed on a chelating resin [J. Porath et al., "Metal Chelate Affinity Chromatography, A New Approach To Protein Fractionation," Nature, 258, pp. 598-99 (1975)].
A difficulty with the technique arises, however, in selectively desorbing the proteins from the resin. A common technique for desorption is lowering the pH to about 3 or 4 [A. J. Fatiadi, "Affinity Chromatography And Metal Chelate Affinity Chromatography," CRC Critical Reviews in Analytical Chemistry, 18, pp. 1-44 (1987)]. Another method consists of adding solutes to the eluent which have a stronger affinity than the proteins for binding to the chelated metal. This involves using strong complexing agents such as histidine or EDTA, which bind tightly to the metal [A. Figueroa et al., "High-Performance Immobilized-Metal Affinity Chromatography Of Proteins On Iminodiacetic Acid Silica-Based Bonded Phases," J. Chromatography, 371, pp. 335-52 (1986)].
With the latter technique, the metal is often stripped from the column; such "bleeding" of the metal ions is obviously an unwanted effect in a purification process. Figueroa et al. reported the use of ammonia, a weak competing ligand, as an eluent to desorb slightly bound proteins from an IMAC column. Their procedure, however, involved the use of HPLC and of a complex binding buffer system, requiring additional washings of the column and switching to ammonia for elution. Both of these factors add to the time involved in running the purification and detract from the efficiency and yield of the purification process.
In addition, the commonly used technique of lowering the pH to desorb proteins from the column is generally effective only for desorption of strongly bound proteins, since low pH desorption often promotes non-selective desorption of all proteins.
Genetic engineering technology has made possible the production of recombinant proteins in quantities hitherto unavailable. However, these proteins often have major contaminants which have presented an obstacle in purifying the proteins into pharmaceutically acceptable form. Current purification procedures are only partially effective in the purification of proteins found with contaminant proteins having similar molecular weight and net charge. As a result there is a continuing need for methods for purifying such proteins so as to increase the availability of new therapeutic agents.